The Probasin (PB) promoter has become the promoter of choice to target genes to epithelial cells in the prostate of transgenic mice, increasingly used in gene therapy constructs to target therapeutic genes to prostate cancer cells, and as an assay to study androgen receptor action. For these reasons, the PB promoter is an ideal model to study the mechanism that controls prostate-specific gene expression. By testing 31 ten base pair linker-mutations of the PB promoter in prostatic and non-prostatic cells and six PB constructs in transgenic mice, we have confirmed that two elements, when mutated, reduced bioactivity only in prostatic cell lines and are required for prostate specific expression in transgenic mice. The two DNA elements, designated as TS 1 and TS2 (TS for Tissue Specific), revealed similar sequences, identical patterns in EMSA and functional interchangeability. Further, TS-1 is immediately upstream and adjacent to the Androgen Receptor Binding Site-1 (ARBS-1) and TS-2 is immediately downstream and adjacent to ARBS-2. We have identified one Transcription Factor (TF) as HNF3tx, a 52 kD forkhead family member. The forkhead family of TFs plays a fundamental role in segment formation, organ development, and cell differentiation. Furthermore, our data demonstrate that HNF3tx functions as a coactivator of AR function. Although over forty coregulators for AR have been identified, neither HNF3alpha nor any other member of theforkhead family ever been implicated in AR action. Our hypothesis is that TS-1 and TS-2 interact with ARBS-1 and ARBS-2, respectively, to form a TF complex that include AR, HNF3alpha, and at least two additional TFs that control prostate-specific expression. Our goal is to identify and characterize the TFs that control prostate-specific gene expression and determine the role of these TFs in prostate development. To test this hypothesis, the three Specific Aims are as follows: I) To determine the mechanism by which HNF3tx regulates PB gene transcription; II) To determine the function of HNF3ct in prostate development; and III) To identify the additional transcription Factors (TFs) that bind in a complex to control prostate-specific gene expression.